Mutational, structural and inhibitory investigations of metallo-β-lactamases involved in antibiotic resistance

Abstract

This thesis focuses on metallo-β-lactamases (MBLs) enzymes that break down a wide variety of antibiotics. Bacteria harboring genes expressing MBLs are antibiotic resistant. If the MBL genes are found on mobile genetic elements, the spread of the genes between bacteria becomes easier. To date, there are no MBL inhibitors available for blocking the enzyme activity, and thus there is an urgent need to find such inhibitors. In order to understand how the MBL enzymes work, information on residues important for the enzyme activity is valuable.

The importance of different residues located in or close to the active site of selected MBLs and the search for MBL inhibitors in synthetically made compounds were investigated. This was performed using enzyme kinetics studies, thermostability measurements, MIC determination, cellular assay, modelling studies, in silico calculations and crystallography.

The substitution of a zinc-binding residue resulted in loss of one active site zinc and the ability for the enzyme to hydrolyze β-lactam antibiotics. Substitutions of second sphere residues contributed in tuning the substrate specificity of the MBL enzymes. Certain second sphere substitutions introduced more hydrogen bonds, which increased the catalytic activity and the temperature stability of the mutants. Other substitutions revealed a reduction in catalytic efficiency, showing that the residues are important, however, not essential for the enzyme activity.

The thiol-based synthesized inhibitors studied in this thesis revealed potent inhibitors with inhibitory effects in the low micro- and nanomolar range. These inhibitors are good starting points for further inhibitors optimization, preferably broad spectrum inhibitors.